Radiochemistry for positron emission tomography

Positron emission tomography (PET) constitutes a functional imaging technique that is harnessed to probe biological processes in vivo. PET imaging has been used to diagnose and monitor the progression of diseases, as well as to facilitate drug development efforts at both preclinical and clinical stages. The wide applications and rapid development of PET have ultimately led to an increasing demand for new methods in radiochemistry, with the aim to expand the scope of synthons amenable for radiolabeling. In this work, we provide an overview of commonly used chemical transformations for the syntheses of PET tracers in all aspects of radiochemistry, thereby highlighting recent breakthrough discoveries and contemporary challenges in the field. We discuss the use of biologicals for PET imaging and highlight general examples of successful probe discoveries for molecular imaging with PET – with a particular focus on translational and scalable radiochemistry concepts that have been entered to clinical use.

Positron emission tomography (PET) constitutes a functional imaging technique that is harnessed to probe biological processes in vivo. PET imaging has been used to diagnose and monitor the progression of diseases, as well as to facilitate drug development efforts at both preclinical and clinical stages. The wide applications and rapid development of PET have ultimately led to an increasing demand for new methods in radiochemistry, with the aim to expand the scope of synthons amenable for radiolabeling. In this work, we provide an overview of commonly used chemical transformations for the syntheses of PET tracers in all aspects of radiochemistry, thereby highlighting recent breakthrough discoveries and contemporary challenges in the field. We discuss the use of biologicals for PET imaging and highlight general examples of successful probe discoveries for molecular imaging with PETwith a particular focus on translational and scalable radiochemistry concepts that have been entered to clinical use.
Translational molecular imaging has facilitated the diagnosis and management of numerous pathologies and has become an integral part of daily clinical routine-with significant implications in oncology, cardiology, and neurology [1][2][3] . In contrast to structural and anatomical imaging, molecular imaging provides functional information via the interaction between a targeted probe and the biological system. In nuclear medicine, the molecular probe constitutes a radionuclide-bearing radiopharmaceutical, frequently referred to as a tracer. From an imaging point of view, the field of nuclear medicine is composed of two mechanistically distinct, albeit somewhat comparable imaging techniques, namely positron emission tomography (PET) and single-photon emission computed tomography (SPECT). While the former imaging modality exploits coincidence detection of colinear annihilation photons, the latter is based on the detection of single gamma rays. Accordingly, although PET and SPECT probes are both generated via the incorporation of a radionuclide within a biomolecule, PET radionuclides emit positrons while SPECT radionuclides are γ-emitters. PET and SPECT are non-invasive techniques that provide information on a molecular and cellular level, which makes them particularly valuable for the detection of biochemical changes at an early stage of disease development, often before clinical symptoms are evident 4 .

Positron Emission Tomography (PET)
Given the excellent sensitivity and the recent technical advances made in accurate image quantification, PET has been widely used for disease staging and drug development purposes [4][5][6][7] . Notably, only trace amounts of the radioactive probe are typically necessary to obtain high-quality images. Due to this remarkable sensitivity, PET radioligands have the capability to probe biological processes without eliciting pharmacological activity 8 . The principle of PET imaging is illustrated in Fig. 1. In the initial step, PET radionuclides are typically generated via nuclear reactions within a cyclotron or by specific decay mechanisms from a generator. The resulting radionuclides are incorporated through a variety of available radiochemical reactions into molecules, thus yielding the desired tracers. Quality control (QC) measures, according to legal requirements and monographs in the respective Pharmacopoeias (e.g., US and European Pharmacopoeia), are undertaken to ensure proper radiopharmaceutical quality for intravenous injection, before the tracer is administered to the patient.
The patient is subsequently scanned in a round-shaped scanner, where quantification of the radioactive signal provides information on the annihilation site, and thereby, the tracer localization at different organs. Importantly, the emitted positron travels a certain distance (positron range), depending on its energy, and undergoes inelastic collisions until the kinetic energy is so low that the timely overlap with an electron and annihilation is possible. Annihilation typically converts the combined mass into electromagnetic energy that can be in the form of two gamma rays released in opposite directions. Spatial image resolution is inherently limited by the positron range of a given PET radionuclide. Coincidences of photons are detected by the full-ring array of detectors of the PET camera and annihilation sources are reconstructed. Indeed, through distinct algorithms, PET data can be reconstructed into the spatial distribution of a given probe. Major challenges in contemporary PET image reconstruction include the correction for scattering and random coincidence events as well as distinct signal attenuation through different body tissues. As such, PET imaging is routinely combined with computed tomography for anatomical orientation and attenuation correction 9 .
Among the various existing positron-emitting nuclides (Table 1), fluorine-18 ( 18 F) is the most widely used radionuclide for PET due to its unique advantages over other PET nuclides. These advantages include (1) the low positron range of <1 mm 10 , which allows the generation of high spatial resolution images, (2) the clear positron emission profile comprising 97% positron emission and 3% electron capture (EC), and (3) the optimal physical half-live of 109.8 min that enables off-site use in satellite facilities without a cyclotron 11 . Notwithstanding the favorable physical properties of 18 F, fluorine atom is typically not a constituent of most biomolecules and hence, frequently introduced via bioisosteric substitution of a hydrogen atom or hydroxyl functionality 5 . These substitutions are not associated with significant steric effects; however, electron-withdrawing fluorine atoms may substantially affect pharmacokinetic and pharmacodynamic properties of PET radioligands 12,13 . Given that carbon atoms are encountered in virtually every biomolecule, carbon-11 ( 11 C) labeling is frequently considered a useful alternative to circumvent the need for bioisosteric substitution, despite the relatively short half-life and higher positron range as compared to 18 F. Indeed, remarkable advances in 11 C-labeling strategies have enabled the labeling of several functional groups, thereby substantially expanding the chemical space of substrates amenable to PET. Besides fluorine-18 and carbon-11, there is a number of other commonly used PET radionuclides (Table 1). In this review, we will summarize commonly used radiolabeling strategies in PET radiochemistry, thereby focusing on recent advances 8,14 with fluorine-18, carbon-11, nitrogen-13, oxygen-15, and other PET radiohalogens, as well as chelator-based radiometal chemistry. Breakthrough examples of  PET ligands with clinical relevance will be discussed. Further, we will elucidate how advances in bioconjugation methods in radiochemistry led to novel opportunities for the use of biologicals in molecular imaging with PET.

PET tracers as diagnostic biomarker and PK/PD tool in drug discovery
Over the past decades, PET imaging has substantially gained attention and is increasingly used not only as a preclinical and clinical research tool but also in routine clinical diagnosis. As such, PET is ideally suited for visualizing molecular and cellular events that occur early in the course of a disease or following therapeutic intervention 9 . Further, depending on the applied tracer, PET can be used to gain prognostic information, offering a valuable risk-stratification tool in clinical practice, as it is currently performed with various probes in atherosclerotic inflammation imaging 2,15 .
In addition to its role in disease diagnosis and therapy monitoring, PET has been established as a vital tool that is being used at various stages of modern drug development. As such, PET can be applied for target validation studies by assessing the target protein expression and localization at different disease stages 6 . Further, lead compound optimization is typically conducted by in vivo assessment of pharmacokinetic and pharmacodynamic drug properties. Proof of target engagement, brain penetration, and potential differences between patients and healthy individuals are of particular interest in PET studies. If amenable to labeling with a PET nuclide, drug candidates can be directly radiolabeled and injected into living organisms, thereby yielding information on drug pharmacokinetics and metabolic stability through blood sampling or post-mortem analysis. This approach is limited by the costly, and in some cases challenging, development of a suitable radiosynthesis for every drug candidate to be tested. As an alternative, promising compounds in development can be challenged by competitive target engagement studies against an established PET probe that shares the same binding site. The advantage of the latter method is that multiple compounds can be effectively screened against one single probe, thereby addressing the following questions: (1) Were the right patients selected to test a given hypothesis and which patient populations are most likely to benefit from this trial (patient stratification)? In many cases, obtaining histological evidence from biopsies is not feasible, whereas a non-invasive assessment of target abundancy in the organ of interest by PET may substantially improve patient selection. (2) Does the drug reach the desired target site and what exposure can be achieved (target occupancy at a given dose)? In addition, while histological assessment may be hampered by protein degradation outside of the human body, and only covers a limited area of the target tissue, PET provides real-time information, allowing whole-body imaging. Obtaining such information at early stage is of paramount value to improving clinical trials and establishing an efficient drug development process. Other applications include the use of PET as accompanying tests for precision therapies, whereas PET probes are frequently employed as basic and clinical research tools to study disease pathophysiology. Indeed, considering the quantitative nature of PET, non-invasive studies to elucidate disease mechanisms and pathophysiology are feasible not only in animal models but also in human subjects, as shown by various recent clinical studies [16][17][18][19] . The translational value of PET is further corroborated by the ability to directly monitor disease progression on a molecular level, thereby providing a unique functional readout that is complementary to structural and morphological information typically detected by conventional imaging modalities.

Translational Radiochemistry
The successful clinical translation of a PET tracer depends not only on its biological characteristics but also on the implementation of practical radiolabeling solutions that allow fully automated tracer synthesis under required quality assurance levels (e.g., GMP, cGRPP). Indeed, the selection of PET nuclide and radiosynthetic approach merits careful considerations since synthetic conditions deemed appropriate for research applications may not be suitable to deliver pharmaceuticalgrade tracers. Potential issues may arise from the lack of precursor stability at ambient temperature, the use of metal catalysts that may exert toxicity if injected into humans, synthetic steps that require manual intervention, volatile solvents with volume reproducibility issues, radioactive and non-radioactive byproducts that co-elute with the tracer, lengthy multistep syntheses that do not yield sufficient radioactivity or insufficient molar radioactivity levels at the end of synthesis, and the lack of tracer stability in the final formulation. The concept translational radiochemistry is introduced herein to emphasize the unique characteristics of PET chemistry aimed at human translation and routine radiopharmaceutical production. To qualify any novel radiochemical method from benchtop discovery as translational radiochemistry, we evaluate it not only by its radiochemical yield and molar activity, but also by the feasibility and translatability of automation or remotely-controlled radiosynthesis in a hot cell, reproducibility and regulatory compliance in a clinical production/ manufacturing environment (e.g., cGMP process) with the operation, participation, and approval of a diverse team, including cyclotron engineers, radiochemists, QC/QA analysts, formulation specialists, radiopharmacists, and nuclear radiologists. As such, translational radiochemistry is refined as scalable, robust, and reproducible PET chemistry that consistently provides high-quality radiopharmaceuticals, meeting a predefined set of specifications for human use, which is critical to protect patients from unnecessary toxicities and ensure the best possible diagnostic care.
When there is no suitable methyl group for 11 C-labeling in the target molecule, alternative 11 C-labeling strategies are employed, such as 11 C-carbonylation. Carbonyl groups are commonly found in biologically active molecules, such as carboxylic acids, aldehydes, ketones, esters, amides, carbamates, and ureas. For [ 11 C]carboxylic acids (Fig. 2b), one classic preparation method is the fixation of [ 11 C]CO 2 with strong basic Grignard or organolithium reagents. A representative example is the preparation of [carbonyl-11 C]WAY100635 26 (a radioligand for imaging 5-HT1A receptor), which has been used for clinical research. The high reactivity of these organometallic reagents limits the potential scope of the method, and these organometallic reagents readily absorb ( 12 C)CO 2 from the atmosphere, thereby leading to low molar activity of the tracer. Transition metal-catalyzed coupling reactions in the presence of [ 11 C]CO and [ 11 C]CO 2 were efficiently employed to introduce [ 11 C]carbonyl groups into various organic compounds. Indeed, Riss and co-workers reported a Cu-mediated carboxylation of boronic acid esters with [ 11 C]CO 2 in 2011 27 , whereas Vasdev, Liang and co-workers used this method in the synthesis of [ 11 C]bexarotene (a retinoid X receptor agonist), with which PET/MR imaging was successfully performed in nonhuman primates 28 . Recently, Gee, Bongarzone and co-workers developed a Cu-mediated carboxylation of trialkoxysilanes and trimethylsilane derivatives with [ 11 C]CO 2 to afford [ 11 C]carboxylic acids 29 . In addition, Karimi and co-workers reported on the Pd-mediated carboxylation of aryl halides/triflates and benzyl halides using [ 11 C]CO in the presence of tetrabutylammonium hydroxide or trimethylphenyl-ammonium hydroxide, respectively 30 . These reactions were conducted in a micro-autoclave at 180-190°C for 5 min using high pressure (35 MPa). Similarly, Telu and co-workers reported a novel Pd-mediated carbonylation of aryl(mesityl)iodonium salts with [ 11 C]CO as an efficient approach to obtain [ 11 C]arylcarboxylic acids in generally good to high yields (up to 71%) 31 . Recently, Lundgren, Rotstein, and co-workers reported the carbon isotope exchange of carboxylic acids via decarboxylation/ 11 C-carboxylation enabled by organic photoredox catalysis 32 . This reaction provides a mild and rapid method for direct carboxylate exchange, but the molar activity is low. For example, [ 11 C]Fenoprofen could be prepared through this method in 9.5% decay-corrected RCY (radiochemical yield 33 , the amount of activity in the product expressed as the percentage (%) of starting activity) with a molar activity of 0.029 GBq/μmol.
For [ 11 C]aldehyde and [ 11 C]ketone ( Fig. 2c), Dahl and co-workers described a Pd-mediated 11 34 . Notably, [ 11 C]formaldehyde proved to be a useful building block in the synthesis of 11 C-labeled compounds via electrophilic aromatic substitution, Mannich-type condensations or cyclization reactions. In 2008, Hooker and co-workers reported a simple, accessible, and high-yielding (up to 89% radiochemical conversion (RCC)) method for the production of [ 11 C]formaldehyde by converting [ 11 C]methyl iodide to [ 11 C]formaldehyde via trimethylamine N-oxide under mild conditions 35 . There are also other methods to access [ 11 C]ketones, such as Pd-mediated 11 C-carbonylative cross-coupling of alkyl/aryl iodides or diaryliodonium salts with organostannanes, aryl iodide or aryl triflates with boronic acids 8 .
[ 11 C]carboxylic acid derivatives (Fig. 3a) [36][37][38] . In 2017, Gee, Bongarzone and co-workers reported the rapid one-pot synthesis of [ 11 C] amides from primary or activated aromatic amines with [ 11 C]CO 2 in the presence of Mitsunobu reagents via an isocyanate intermediate, followed by the addition of Grignard reagents 39 . The respective [ 11 C] amides were generated in moderate RCYs, including [ 11 C]melatonin (a hormone of the pineal gland), which was obtained in 36% RCY and with a molar activity of 70-100 GBq/µmol. Transition metal-mediated carbonylation cross-coupling reactions with [ 11 C]CO provide an alternative pathway to afford a variety of 11 C-labeled compounds. The main challenge is the low solubility of [ 11 C]CO in organic solvents. Indeed, several strategies have been introduced to overcome this limitation, including [ 11 C]CO recirculation, high-pressure reactors, continuousflow microreactors, xenon as carrier gas (the high solubility of xenon gas in organic solvents facilitates the transfer of [ 11 C]CO), [ 11 C]COreleasing molecules (such as silacarboxylic acids), solvent-soluble adducts of [ 11 C]CO (such as BH 3 ·[ 11 C]CO), and the addition of XantPhos -a hindered bidentate phosphine ligand, found to facilitate the 11 C-carbonylation process. Among these improved strategies, Skrydstrup, Antoni and co-workers developed pre-generated Pd-aryl oxidative addition complexes as stoichiometric reagents in carbonylation reactions with [ 11 C]CO to produce structurally challenging, yet pharmaceutically relevant compounds, including [ 11 C]raclopride (a dopamine D2/D3 receptor antagonist), which was obtained in 38-44% RCY (decay-corrected) and with a molar activity of 333-407 GBq/ μmol 40 . The use of pre-generated Pd-aryl complexes were an excellent solution to utilize the oxidative addition process in 11 C-carbonylation of structurally challenging substrates. The Pd-mediated 11 C-carbonylation reactions provide an alternative access to PET tracers, but the electrophiles used in this reaction were limited to aryl, alkenyl, methyl, or benzyl halides and triflates without β-hydride to  avoid the competing β-hydride elimination. In 2016, Rahman and coworkers overcame the limitation and reported the nickel-mediated 11 C-aminocarbonylation of non-activated alkyl iodides containing βhydrogen with [ 11 C]CO at ambient pressure 41 . Ni(COD) 2 and bathophenanthroline were used as pre-catalyst and ligand, respectively. Several aliphatic [ 11 C]amides were furnished via this method in moderate to good yields. Transition metal-mediated 11 C-carbonylation was also used to prepare [ 11 C]esters (cf. recent 11 C-carbonylation review 42 ); however, the reaction scope was often limited to substrates lacking βelimination pathway. One approach to extend the scope of accessible labeled esters entails photoinitiated free radical reactions by Långström and co-workers in the synthesis of [ 11 C]carboxylic esters from a variety of primary, secondary, and tertiary alkyl iodides and [ 11 C]CO 43 53 . [ 11 C]COF 2 was prepared in quantitative yields by passing [ 11 C]CO gas through a AgF 2 column at room temperature and then it was employed directly in the synthesis of a wide range of [ 11 C] heterocycles, including [ 11 C](S)-CGP-12177 (a β-adrenoceptor radioligand) and [ 11 C]DMO (a radiotracer for determining tissue pH in vivo).
As nitriles are often found in biologically active molecules and are also used in the synthesis of carboxylic acids, amides, amines, and related derivatives, several 11 C-cyanation methods have been developed (Fig. 3c). As such, aliphatic [ 11 C]nitriles can be obtained by nucleophilic substitution with [ 11 C]cyanide ions. The transition-metalmediated aromatic 11 C-cyanation reaction is an attractive approach to prepare aryl [ 11 C]nitriles and has been investigated for more than 20 years. The limitation of these reactions often entails harsh reaction conditions, such as high temperatures, relatively long reaction times, and the use of inorganic bases, which limits the substrate scope. In 2015, Hooker, Buchwald and co-workers reported a rapid Pd-mediated 11 C-cyanation of aryl halides or triflates with [ 11 C]HCN and biaryl phosphine as a ligand at room temperature 54 . The sterically hindered biaryl phosphine ligands facilitated a rapid transmetalation with [ 11 C]HCN and reductive elimination of aryl nitriles, whereby the reactions were completed in 1 min. A range of aryl [ 11 C]nitriles was prepared via this method, including several drugs, such as [ 11 C]perampanel (antiepileptic drug) obtained in 21% RCY (decay-corrected) with a molar activity of 50 GBq/μmol, and [ 11 C]citalopram (antidepressant) obtained in 85% RCC. Recently, Hosoya, Zhang and co-workers developed a Pd-mediated 11 C-cyanation of (hetero)arylboronic acids or esters with [ 11 C]NH 4 CN/NH 3 in high RCYs 55 . This method demonstrated excellent functional group tolerance and wide substrate scope. In addition, Cu-mediated 11 C-cyanation provided an alternative access to aryl [ 11 C]nitriles. Initially, Ponchant and co-workers developed the Cu-mediated 11 C-cyanation of aryl halides, where [ 11 C]cyanide ions were trapped by copper at high temperatures (180°C) 56 . In 2017, Liang, Vasdev and co-workers developed the first Cu-mediated 11 C-cyanation of aryl boronic acids in aqueous solutions 57 . Indeed, a broad range of arylboronic acids was labeled in 9-70% RCYs via this method. In the following year, Scott, Sanford, and co-workers further extended Cumediated 11 C-cyanations to arylstannanes and other arylboron precursors with Cu(OTf) 2 58 . Trifluoromethyl (CF 3 ) groups can be found in many drugs and potential PET radiotracers. An efficient method to incorporate 11 C-labeled trifluoromethyl group ([ 11 C]CF 3 ) with high molar activity is necessary (Fig. 3d). In 2017, Pike and co-workers reported the synthesis of [CF 3 -11 C]trifluoromethylarenes from arylboronic acids, aryl iodides and aryldiazonium salts with [ 11 C]CuCF 3 59 . [ 11 C]CuCF 3 was prepared from [ 11 C]fluoroform with copper(I) bromide and potassium tert-butoxide, while [ 11 C]fluoroform was generated from cyclotron-produced [ 11 C]methane passing over heated (270°C) cobalt(III) fluoride (CoF 3 ) in good yield. As [ 11 C]methane can be generated by cyclotron with a high molar activity, a range of [CF 3 -11 C]trifluoromethylarenes could be synthesized via this method with a high molar activity (200−500 GBq/ μmol), such as flutamide (an antiandrogen drug) obtained in 76% RCY (decay-corrected) and leflunomide (an antirheumatic drug) obtained in 93% RCY (decay-corrected) with a molar activity of 400 GBq/μmol. Therefore, [CF 3 -11 C]-trifluoromethylation with [ 11 C]CuCF 3 from [ 11 C]fluoroform represents an important breakthrough for labeling PET tracers with trifluoromethyl groups with high molar activity.
Nitrogen-13 and Oxygen-15. Nitrogen-13 ( 13 N, half-life t 1/2 = 10 min) and Oxygen-15 ( 15 O, half-life t 1/2 = 2 min) are short-lived PET radionuclides, rendering them unsuitable for multiple-step radiosynthesis in most scenarios 8 . Of note, [ 13 N]NH 3 is the most important 13 N-labeled tracer and it could be produced via the 16 O(p,α) 13 N nuclear reaction 60 . Currently, [ 13 N]NH 3 has been widely used in clinical PET imaging to identify myocardial perfusion defects and assess coronary flow reserve in patients with suspected coronary artery disease. In addition, [ 13 N]NH 3 serves as a building block for further 13 18 F-fluorination is conducted in polar aprotic solvents, such as MeCN, DMF, or DMSO, to pursue high 18 F-incorporation in a relatively short reaction time (usually ≤30 min). In a counterintuitive manner, Chi and co-workers reported an effective nucleophilic 18 F-fluorination of aliphatic mesylates in ionic liquid and protic tert-butyl alcohol 63 . These reactions showed an increased 18 F-fluorination rate and a decreased formation of undesired elimination byproducts. 18 F-Fluorination of alcohols commonly involves two steps, including the modification of the hydroxyl group (-OH) to a leaving group (-OTs, -OMs, or -ONs) and the subsequent 18 F-fluorination. In 2015, Doyle and co-workers reported PyFluor (2-pyridinesulfonyl fluoride) as a deoxyfluorination reagent for alcohols 64  Transition metal-mediated aliphatic 18 F-fluorination provides mild conditions to access alkyl [ 18 F]fluorides. As such, Gouverneur, and Nguyen and co-workers successfully developed Pd-and Ir-mediated allyl 18 F-fluorination reactions [66][67][68] . Further, Groves, Hooker and coworkers reported the Mn-mediated 18 F-fluorination of benzylic C−H and non-activated secondary and tertiary C−H bonds 69,70 . Doyle and co-workers developed the Co-mediated enantioselective aliphatic 18 F-fluorination of epoxides 71 .
In particular, 18 F-fluorination involved reductive elimination of the C − 18 F bond from a palladium(IV) aryl [ 18 F]fluoride complex intermediate. Subsequently, Ritter and co-workers described a nickel-mediated oxidative radiofluorination approach with a hypervalent iodine oxidant at room temperature, requiring <1 min reaction time 85 . In 2014, Gouverneur and co-workers described a Cu-mediated 18 F-fluorination of aryl boronic esters (aryl-BPin) (Fig. 4b-5) 86 . As aryl-BPin reagents have high stability to air and moisture, they are considered excellent precursors for 18 F-fluorination. Indeed, the latter approach was used to radiofluorinate a large range of electron-neutral, -rich, and -poor arenes, including 18 F-gefitinib, obtained in 22% RCC (an inhibitor of the epidermal growth factor receptor−tyrosine kinase), and [ 18 F]flutemetamol (a β-amyloid-targeted probe) obtained in 9% RCC, and clinically validated [ 18 F]FDOPA obtained in 10% RCY (decay-corrected) with a molar activity of 76 GBq/μmol 87 . Subsequently, Scott, Sanford and coworkers also reported Cu-mediated 18 F-fluorination of aryl boronic esters (aryl-BPin) and aryl boronic acids 88 . Additionally, diaryliodonium salts 89 and aryl stannanes 90 were found to be useful precursors for Cu-mediated 18 F-fluorination, and a range of clinically validated PET tracers was prepared via these methods, such as [ 18 F]SDM-8 (a PET tracer for imaging synaptic density) was obtained from 18 F-labeling of aryl stannane precursor in 35% RCY (decay-corrected) with a molar activity of 241.7 GBq/μmol 91 . Recently, Li, Nicewicz and co-workers developed the aryl C−H 18 F-fluorination via organic photoredox catalysis (Fig. 4b-6) 92 . An organic acridinium-based photocatalyst was used, along with TEMPO as a redox co-mediator. Various electron-rich aromatics were 18  As an electrophilic 18 F-fluorination reagent, [ 18 F]F 2 can be used as an alternative F-18 source (Fig. 5a). With non-radioactive F 2 applied as a carrier gas, the production of [ 18 F]F 2 typically yields low molar activities (0.04 to 0.4 GBq/μmol), which limits its widespread use. To overcome this limitation, an improved method was developed, harnessing the gas phase 19 96 . Similarly, [ 18 F]Selectfluor was developed by Gouverneur, Luthra, Solin and co-workers and utilized for the 18 F-fluorination of silyl enol ether and electron-rich arylstannanes 97 -including the synthesis of 6-[ 18 F]FDOPA. However, these approaches still yield tracers with low molar activities, and further work has to be conducted to extend this method for clinically relevant tracer production.
In addition, a number of 18 F-labeling methods via B-, Si-, Al-, S-18 F bond formation were developed in the past decade and reviewed recently (Fig. 5c) 8,108,109 . As B-, Si-, and Al-18 F bonds have high bond dissociation energies, the 18 F-labeling process is efficient in an aqueous solution, which is highly suited for labeling biomolecules for PET imaging studies. Although low pH and/or elevated temperatures are still needed for efficient 18 F-labeling, a recent advance has been made to achieve Al-18 F efficient labeling at physiological temperatures 110 . Compared to the C-18 F formation via S N 2-or S N Ar-type reaction, S IV -18 F formation has a lower kinetic barrier, thus enabling 18 F-labeling at room temperature as evidenced in the work of aryl [ 18 F]fluorosulfates by Wu, Yang, Sharpless and co-workers, including a PARP1-targeted probe for tumor imaging 111 .
Furthermore, a stepwise and indirect 18 F-labeling method via the link 18 F-labeled prosthetic group to the target molecule is an attractive approach for labeling well-functionalized molecules 109  . More details about the 18 F-labeled prosthetic groups and strategies to increase the metabolic stability of radiotracers can be found in some excellent reviews [113][114][115] .

Related radiohalogens (Bromine-76 and Iodine-124 chemistry).
Iodine-124 is usually produced via 124 Te(p,n) 124 I nuclear reaction in the cyclotron and Iodine-124 ( 124 I, t 1/2 = 4.2 d) has a relatively long half-life of 4.2 days, which is attractive for investigation of enduring biological processes in vivo. Although the maximum positron energy of 2.14 MeV and the positron intensity of 25% make iodine-124 not an ideal positron emitter, iodine-124 is still a clinically relevant long-lived, nonradiometal nuclide, which enables quantitative PET imaging over several days. The well-established iodine chemistry provides an excellent platform to incorporate 124 I into organic compounds. Generally, nucleophilic 124 I-iodination reactions can be used to synthesize 124 I-labeled alkyl or aryl compounds via S N 2 or S N Ar mechanism (Fig. 6). For example, the hypoxia imaging agent [ 124 I]IAZA could be generated via a nucleophilic substitution reaction with sodium [ 124 I]iodide 116 . In recent years, copper (I) salts were found to be an accelerant for halogen- 124 118 , and morpholino-[ 124 I]IPQA (EGFR kinase PET tracer) obtained in 14% RCY 119 . In addition, electrophilic 124 I-iodination has also been used in labeling biomolecules, like peptides, antibodies, and proteins, and the presence of tyrosine, histidine, or an aromatic moiety is needed to complete such transformation. Notably, if the pH exceeds 8.5, the 124 I-iodination of histidine is preferred 120 .
Bromine-76 is often produced via 76 Se(p,n) 76 Br nuclear reaction in the cyclotron and bromine-76 ( 76 Br, t 1/2 = 16.2 h) has a medium half-life of 16.2 h, longer than fluorine-18 (t 1/2 = 109.8 min) and shorter than iodine-124 (t 1/2 = 4.2 d). Bromine-76 has a high positron energy (4 MeV) and its positron emission intensity is 57%. Similar to 124 I-iodination reactions, bromine-76 could also be introduced into organic molecules via nucleophilic 76 76 Brbromination of the corresponding tin precursor in the present of peracetic acid in 51% RCY, and this tracer was evaluated in PET imaging of mice with brain tumor 121 . Recently, Zhou and co-workers reported copper-mediated 76 Br-bromination of aryl boron precursors, and a 76 Br-labeled derivative of Olaparib (an inhibitor of PARP-1 (poly (ADPribose) polymerase-1)) was synthesized in 99% RCC via this method 122 .
Clinical examples of small molecule-based PET pharmaceuticals. In this chapter, we will discuss selected examples of PET radioligands that have obtained US Food and Drug Administration (FDA) approval, thereby elaborating on their clinical indications as well as their impact on patient management in clinical routine (Fig. 7). While PET radiopharmaceuticals are of paramount value in both diagnostic imaging and drug discovery 123 , the majority of contemporary FDA-approved small molecule PET radiopharmaceuticals are labeled with fluorine-18, which is attributed to its optimal decay properties, including a relatively clean positron decay, a short positron range as well as a physical half-life that allows satellite distribution 5   been widely used for patient diagnosis, disease staging, and therapy monitoring of various pathologies in oncology, neurology, and cardiology.
Although [ 18 F]FDG remains the most widely used PET tracer in the clinic, there is a continuous demand for more specific probes that are highly selective for a given biological target. As such, over the past several decades, PET has provided unique and powerful insights into normal brain function as well as various neurological disorders and neurodegenerative diseases via probes that accumulate at the sites of misfolded protein aggregates. For instance, Alzheimer's disease (AD) constitutes a progressive neurodegenerative disease that is typically characterized by extracellular amyloid-beta (Aβ) plaques and intracellular neurofibrillary tangles that are formed by misfolded tau aggregates 124 . Notably, the ground-breaking discovery of Aβtargeted PET ligand, [ 11 C]PIB, and its successful application for the early diagnosis of AD fueled the development of radiofluorinated analogs, [ 126 .
In cardiology, PET imaging is a powerful tool for in vivo evaluation of myocardial function. Among other PET probes, [ 13 N]NH 3 was approved by the FDA for rest/stress myocardial perfusion imaging (MPI) to detect ischemic heart disease. The strength of PET-MPI lies in the ability to measure absolute myocardial blood flow at rest and following pharmacologically induced stress, which allows the assessment of perfusion defects in larger coronary arteries, as well as the calculation of coronary flow reserve-an indicator of microvascular function. Notably, PET has been established as the clinical reference standard for the quantification of myocardial perfusion 127 .
In oncology, non-invasive tumor imaging provides crucial advantages in the assessment of tumor progression and regression. In 2020, [ 18 F]fluoroestradiol was approved by the FDA for imaging estrogen receptor (ER)-positive breast cancer lesions 128 . Prostate cancer is common cancer among men, and PET imaging is a powerful tool for monitoring the progression or recurrence of prostate cancer 129 . Indeed, [ 11 C]choline was the first FDA-approved PET tracer that was employed for the detection of recurrent prostate cancer. Similarly, [ 18 F]fluciclovine, an 18 F-labeled analog of L-leucine, was approved by the FDA for imaging prostate cancer recurrence. Although these probes proved to be useful in prostate cancer patients, their accumulation reflected the increased amino acid turnover of tumor cells; however, their mechanism of uptake was not related to tumor-specific markers. In contrast, the more recently FDA-approved prostate-specific membrane antigen (PSMA)-targeted PET radioligand, [ 18 F]DCFPyL (Pylarify®), proved to be highly selective for PSMA-positive tumor cells and as such, provide high accuracy for the detection of PSMA-positive lesions in men with prostate cancer.
PET chemistry of radiometals PET Radiometals. Besides organic PET radionuclides, radiometals provide new possibilities to radiolabel targeted vectors for PET which typically consists of biological molecules (e.g., peptides, proteins, and antibodies). Metal-based radiopharmaceuticals (Fig. 8a) often include a radiometal (M*) bound to a chelator (C), which is attached to a targeting vector (V) through a linker (L) in the format of M*-C-L-V. Compared to other PET radionuclides, radiometals generally have a straightforward labeling process that relies on coordination chemistry. Further, the relatively mild labeling conditions make radiometals suitable for labeling complex biological molecules. Notably, PET radiometals have a wide range of physical half-lives, ranging from hours to several days, such as gallium-68 ( 68 Ga, t 1/2 = 67.7 min), copper-64 ( 64 Cu, t 1/2 = 12.7 h), and zirconium-89 ( 89 Zr, t 1/2 = 78.4 h), which provides a broad radionuclide scope with suitable physical half-life to match the intended application. As such, short-lived radiometals can be used for vectors with fast pharmacokinetics and long-lived radiometals to visualize long-lasting biological processes.
An increasingly utilized feature of PET imaging is radiotheranostics. When a radiometal-based tracer is used in PET imaging for diagnosis, the following radiotherapy could be conducted by changing M* from a positron emitter ( 44 Sc, 64 Cu, 68 Ga, or 89 Zr) to an alpha emitter ( 225 Ac, 212 Pb, or 213 Bi) 130,131 or beta emitter ( 177 Lu, 90 Y, or 67 Cu) for radiotherapy 132 . Theranostic pairs are also feasible without radiometals, for example, with Iodine isotopes 133 135 , but the inherent potential in the theranostic approach with radiometals and chelators is more modular. Of note, contemporary concepts and potential future developments in radiotheranostics have been recently reviewed by Weissleder and co-workers 133 . We will focus on the commonly used radiometals in PET, including 68 Ga, 64 Cu, and 89 Zr. Other radiometals are discussed elsewhere 136,137 . 68 Ga (t 1/2 = 67.7 min) can be produced by commercially available generators ( 68 Ge/ 68 Ga) and is easily accessible to radiochemists. Owing to the long half-life of the parent isotope 68 Ge (t 1/2 = 271 days), 68 Ge/ 68 Ga generators can typically be used within one year. The different chemical properties between 68 Ga and 68 Ge make the separation possible. In the past ten years, several excellent 68 Ga-based radiopharmaceuticals have been developed for clinical use. The accessibility and potential of these radiopharmaceuticals make 68 Ga attractive and promising in PET. It should be noted, however, that the limited capacity and supply of GMP-grade 68 Ge/ 68 Ga generators has prompted the search for improved ways to generate gallium-68 via cyclotron. As an important improvement and supplement to the generator method (usually at ≤100 mCi capacity), the cyclotron-based method could produce 68 Ga in high quantity up to multi Ci levels, which provides strong support for the successful use of 68 Ga in the clinic, especially for a larger patient population 138 . 64 Cu can be produced by different methods via a reactor, an accelerator or a cyclotron. Highly enriched 64 Ni is used in the 64 Ni(p,n) 64 Cu nuclear reaction within a cyclotron to produce carrier-free 64 Cu in high purity, yield, and molar activity 139 . Unlike other PET radiometals, [ 64 Cu]Cu 2+ is not redox-inert under physiological conditions. Indeed, [ 64 Cu]Cu 2+ can be reduced to [ 64 Cu] Cu + under reducing conditions in vivo, which represents a practical consideration during PET tracer design and/or chelator selection (vide infra). In certain cases, the redox dynamics of copper offer some advantages. For example, this redox behavior is the mode of action for [ 64 Cu]Cu-ATSM 140 , a hypoxia imaging tracer. In hypoxic regions, Cu(II) is reduced and subsequently trapped, which can be visualized via PET. Of note, the smaller positron range of 64 Cu compared to 68 Ga renders tracers based on 64 Cu applicable for pathologies with higher spatial requirements, such as cardiovascular diseases. 89 Zr is typically produced via the 89 Y(p,n) 89 Zr nuclear reaction in a cyclotron. With a relatively long half-life (t 1/2 = 78.4 h), 89 Zr can be transported to nuclear medicine facilities without an on-site cyclotron and can be used in antibody-based PET imaging (immuno-PET). Recently, 44 Sc has gained attention as a promising PET radionuclide due to its clean β + -decay (94%) and suitable physical half-life (t 1/2 = 4 h). 44 Sc can be produced either via 44 Ca(p,n) 44 Sc in a cyclotron or by 44 Ti/ 44 Sc generators.
Chelators. Contrary to the labeling with other PET radionuclides involving covalent bond formation (e.g., 18 F-C bond), labeling with radiometals requires coordination with chelators (Fig. 8b). Even if certain chelators prefer some radiometals over others, no chelator is fully metal-specific and will bind other non-radioactive metals, if present during radiolabelling. Chelators are essential to ensure (1) fast complexation of the radiometal at low temperatures, low substrate concentrations, and physiological pH, and (2) thermodynamic and kinetic stabilization of radiometals by forming inert complexes. In general, chelators can be classified in three types, acyclic chelators, cyclic chelators, and cross-bridged chelators.
Acyclic chelators provide fast complexation kinetics at room temperature and physiological pH. Nonetheless, acyclic chelator-metal complexes, in general, exhibit less favorable stability than the cyclic ones. The earliest chelators for radiometals were acyclic, such as the polycarboxylate chelators EDTA and DTPA. Although these chelators exhibited fast complexation kinetics at room temperature, the complexes were plagued by their low stability in vivo. Further, EDTA and DTPA have low selectivity for cations with a similar valence; however, they bind M 3+ with a higher affinity than M 2+ . The acyclic chelator HBED-CC is unique in its use of two lipophilic phenolic groups for [ 68 Ga]Ga 3+ coordination 141 . The [ 68 Ga]Ga-HBED-CC complex consists of two negatively charged carboxyl groups, two neutral nitrogen atoms and two negatively charged phenolic oxygens, creating a complex with one negative charge and with a more hydrophobic character (from the benzyl rings of the phenolic groups), as compared to other acyclic chelators. The acyclic chelator desferoxamine B (DFO) 142 has been developed and recognized as a gold standard chelator for Zr 4+ in the past decade. DFO is an iron-binding siderophore from the bacteria Streptomyces pilosus, which was approved by FDA for iron-overload disease, using the six oxygens of three hydroxamate groups to bind Zr 4+ in a [ 89 Zr][Zr(DFO)(H 2 O) 2 ] + complex. Escorcia and co-workers recently showed that [ 89 Zr]Zr-DFO-onartuzumab, a monoclonal antibody targeting the Met receptor tyrosine kinase, can be used to predict the therapeutic response to targeted radioligand therapy in a rodent model of pancreatic cancer that was resistant to Met kinase inhibitors 143 . With the long biological and physical half-lives of [ 89 Zr]Zr-DFO-mAbs (mAb, monoclonal antibody), the chelation stability of [ 89 Zr]Zr-DFO-mAb needs improvement. Hence, there is an ongoing effort to obtain 89 Zr chelates with improved stability. Notably, Mindt, Gasser, and co-workers developed a novel chelator DFO* by adding one more hydroxamate donor to DFO, and the resulting two more coordination sites of DFO* largely improved the stability of [ 89 Zr]Zr-DFO* complexes 144 .
To improve the stability of M*-C complexes, cyclic chelators have been designed to strengthen the binding of radiometals. Cyclic chelators have more rigid chemical structures, in which the donor atoms are in a more optimal position to chelate certain radiometals. Cyclic complexes are generally more stable than acyclic complexes. Although many chelators react with radiometals at low temperatures, albeit at lower radiochemical yields, higher temperatures (40-100°C) are typically needed for rapid radiometal binding. A few exceptions can also be found in the cases of [ 64 110,145 , which is advantageous for biologics that are sensitive to elevated temperatures (>37°C).
Cross-bridged cyclic chelators (Fig. 8b) have been developed to provide additional rigidity over their cyclic counterparts of similar ring dimensions, in most cases cross-bridging between N atoms and reducing the number of carboxyl groups available for radiometal binding. In general, the complexation process requires higher temperatures than cyclic chelators. [ 64 Cu]Cu-CB-DO2A and [ 64 Cu]Cu-CB-TE2A were found to have superior in vivo stabilities to DOTA and TETA. Cross-bridged chelators are widely used with [ 64 Cu]Cu 2+ , possibly owing to the aforementioned redox potential of [ 64 Cu]Cu 2+ , requiring more stable metal-chelate complexes to ensure continued binding, even on reduction. In addition, DiAmSar is a sarcophagine type chelator that can be labeled with [ 64 Cu]Cu 2+ at room temperature. The DiAmSar chelator shows an even higher in vivo stability than most other cross-bridge cyclic chelators 146 . Another sarcophagine analog, MeCOSAR 147 , was used in the synthesis of [ 64 Cu]Cu-SARTATE in the clinical trial (NCT04438304) for imaging patients with known or suspected neuroendocrine tumors. Other chelators for radiometal labeling include, but are not limited to, DOTA variants, NOTA variants, TE2A variants, PCTA, macropa, HOPO, sulfur-containing chelators (e.g., ECD), DATA analogs, DTPA analogs (e.g., CHX-A"-DTPA), octapa and etc. A comprehensive discussion of these chelators is beyond the scope of the present review and can be found elsewhere 136,146,148 .
Linkers. Linkers are important for radiometal-based radiopharmaceuticals, as a direct coupling of the chelator and vector may result in the reduction of receptor affinity. To address this problem, a linker can be introduced between the chelator and the vector. Further, the linker can be utilized to fine-tune the physical properties of the tracer to affect in vivo behavior. An ideal linker does not negatively affect the vector affinity towards the target (sometimes even increases the binding affinity) but can be used to improve the pharmacokinetic properties of the tracer. While linkers have not been studied to a greater extent for radiopharmaceuticals, and the reports are not conclusive, the lessons we learned from antibody-drug-conjugates demonstrated that the appropriate linker depends highly on vector and chelator 149 .
Properties of linkers are critical because a suitable linker may improve radiotracer pharmacokinetics properties. There are several commonly used linkers, such as short sequences of amino acids, polyethylene glycols (PEG), and hydrocarbon chains (Fig. 8c). Simple hydrocarbon chains tend to increase the lipophilicity of radiotracer with an unfavorably slow clearance via the hepatobiliary pathway to follow. In contrast, linkers of amino acid sequences tend to increase the hydrophilicity of radiotracer with primary excretion via the renalurinary pathway as a consequence. Generally, a radiotracer needs to achieve a high target accumulation and a high target-to-background ratio in the shortest possible time. This means that the radiotracer needs to reach the target and be cleared from non-target tissues quickly, while still matching the binding kinetics of the vector. The introduction of PEG linkers to the radiotracers was found to increase receptor-mediated uptake and renal-urinary clearance, decrease nonspecific binding, and achieve a high target-to-background ratio, in most cases. As an example, the introduction of PEG linker in [ 64 Cu]Cu-DOTA-PEGn-AVP04-50 could significantly decrease kidney uptake, and increase tumor uptake, compared to the non-PEGylated counterpart 150 . And for [ 64 Cu]Cu-NOTA-PEGn-trastuzumab 151 , the clearance of PEGylated-radiotracer was three-fold faster while maintaining comparable tumor uptake compared to its non-PEG counterpart. In addition, some functional moieties, including albumin binders, can be introduced to the linker to improve the drug pharmacokinetics. For example, Evans Blue has been introduced to the linker to help the drugs' binding to albumin, extend the half-life, and decrease renal clearance 152 . In all, the interplay of vector, linker, and radiometalchelator determines in vivo imaging properties of the tracer. While the radiometal, chelator and vector are often pre-determined, the linker is easier to modify at late stage, making it an ideal component for finetuning and achieving optimal physicochemical properties of radiometal-based pharmaceuticals.
Clinical examples of radiometal-based PET pharmaceuticals. Although 99m Tc-labeled radiopharmaceuticals have historically dominated the landscape of radiometal-based clinical applications, the enormous potential of radiometals in nuclear medicine goes well beyond these conventional probes. Indeed, the clinical use of gallium-68 has experienced remarkable growth with recent examples of successful phase III clinical trials-particularly in oncology 133,153 . The representative examples include somatostatin receptors (SSTRs)-targeted probes in neuroendocrine tumors and PSMA-targeted probes in prostate cancer (Fig. 8d). Even though there are many reports on antibodies labeled with 89 Zr or 64 Cu, some of which are currently in clinical trials, none of these has been FDA approved yet.
SSTRs are receptors for somatostatin, a small neuropeptide associated with neural signaling. Overexpression of SSTRs on the cell membrane is a unique feature of neuroendocrine tumors, which is a promising target for imaging diagnosis and radiotherapy of neuroendocrine tumors. In the past decade, a range of somatostatin analogs have been developed for SSTRs PET imaging and targeted radiotherapy of neuroendocrine tumors, and the commonly used tracers include [ 68  PSMA has a very low expression in normal prostate cells, but a high expression in prostate cancer cells, and the expression level is directly proportional to prostate cancer progression. Hence, PSMA is a promising target for imaging diagnostics and targeted radionuclide therapy for prostate cancer and its metastases. Currently, various PET tracers have been developed for the diagnosis and treatment of prostate cancer, such as [ 68 46 have not yet been approved by the FDA, the results obtained so far have been promising.
More recent examples of radiometal-based PET pharmaceuticals that were advanced to human research include the angiotensinconverting enzyme 2 (ACE2)-targeted probes, [ 64 Cu]HZ20, and [ 68 Ga] HZ20 164 . Notably, ACE2 constitutes a key viral gateway that facilitates SARS-CoV-2 entry into host cells [165][166][167] . As such, it was hypothesized that non-invasive quantification on ACE2 abundancy in the lungs and other organs holds promise to deliver prognostic value and support the development of ACE2-targeted therapeutic intervention 164 168 . PET imaging with [ 89 Zr]Zr-Atezolizumab before and after the therapy might be of great usefulness for determining PDL-1 expression in tumors.

Bioconjugation in PET tracer development
Herein, we refer to bioconjugation as a general chemical process to form a covalent bond between two molecules, of which at least one is a biomolecule (can be found in biological systems). Of note, biomolecules such as antibodies, antibody fragments and peptides have become a crucial component of modern drug development. The continuously growing number of FDA approvals for therapeutic biomolecules has prompted enthusiasm to further exploit the use of biomolecules for diagnostic purposes 169,170 . While PET radiometals have traditionally been used in combination with antibodies, novel radiochemical tools have widened the radiolabeling scope, particularly with regard to non-metal radionuclides 171,172 . Bioconjugation reactions can be subdivided into conventional methods, that use primary amine with N-hydroxysuccinimides (NHS), isothiocyanates and anhydrides as well as thiol modifications with maleimides, and modern click-type bioconjugation methods, which are typically based on orthogonal reactions including, but not limited to, azide-alkyne cycloadditions, Staudinger ligations and inverse electron demand Diels-Alder cycloadditions (Fig. 9) 173 . Orthogonal reactions are highly selective between a pair of chemical functionalities and can proceed with high yields in aqueous media and at ambient temperature 174,175 . The employed chemical functionalities are typically not present in nature, which allows specific reactions in the presence of naturally occurring functional groups. When orthogonal reactions are inert to biological moieties, they are termed bioorthogonal. Indeed, bioorthogonal reactions constitute the cornerstone of pretargeted imaging, where two components, namely the functionalized targeting vector and the radioisotope-bearing prosthetic group, are administered separately and react in vivo 176 . In this chapter, we will discuss conventional and modern click-type bioconjugation methods, thereby highlighting recent advances in orthogonal PET chemistry for fast and efficient in vitro/in vivo applications.
Conventional bioconjugation. Conventional bioconjugation methods harness the reactivity of amines and thiols, which are present in natural amino acids such as lysine and cysteine residues as well as the Nterminal residues (Fig. 9a). Traditionally, the radiolabeling of proteins was accomplished by reacting their amine or thiol functionalities to chelator-bearing reactive electrophiles and subsequent chelation of the radionuclide, or reaction with radiolabelled prosthetic groups 148 . Indeed, the vast majority of traditional bioconjugation examples involve the reaction of cysteine residues with radiolabeled maleimides via Michael addition or the modification of primary amines with radiolabeled NHS esters, isothiocyanates and activated carboxylic acid derivatives [177][178][179][180][181] . Furthermore, these reactions are mainly not sitespecific since biomolecules such as antibodies contain multiple primary amines (lysines), resulting in the difficulty of controlling bioconjugate sites, potentially impacting their binding properties. Despite their widespread use, conventional bioconjugation reactions offer room for improvement, particularly with regard to reaction kinetics, radiochemical yields and stability issue with maleimide adducts (via retro-Michael addition) 173 .
Modern click-type bioconjugation. In the past, the vast majority of bioconjugation concepts have relied on conventional bioconjugation reactions. To overcome the previously mentioned limitation of conventional bioconjugation methods, however, radiochemists have increasingly focused on labeling strategies that involve orthogonal clickable prosthetic groups (Fig. 9b). Indeed, a variety of click-type bioconjugation methods have been developed, and in some cases, provided more optimal reaction kinetics, higher radiochemical yields, or more stable labeled vectors. Orthogonal reactions that rely on naturally occurring primary amines do not provide site-selective radiolabeling since biomolecules typically bear multiple primary amines. Nonetheless, the high chemical selectivity of orthogonal reactions provides a more uniform tracer, thereby easing a subsequent regulatory process 173 . Among the orthogonal reactions harnessed by radiochemists, azide-alkyne cycloadditions (AACs) have been most extensively employed for peptide and protein labeling [182][183][184][185][186][187][188][189][190][191][192][193][194] . A unique advantage of this reaction is that the click product, a 1,2,3-triazole ring, is well tolerated by the binding domain due to its relatively small and rigid structure, which essentially forms the bioisostere of an amide linkage. In light of the highly suited AAC properties for fast and efficient radiolabeling, a wide variety of radiofluorinated alkyne-or azidebearing prosthetic groups were harnessed with great success, yielding numerous radiolabeled vectors including peptides, oligonucleotides, and proteins [195][196][197][198][199][200][201][202][203][204][205] .
Notwithstanding the successful implementation of CuAAC in PET radiochemistry, this reaction has some shortcomings, namely, the need for a copper catalyst and the presence of a reductant (commonly ascorbic acid), both of which may interact or react with the vector as well as the radiolabeled prosthetic group. Particularly, serine, histidine, and arginine residues can coordinate copper cations, imposing potential structural and functional changes on the vector 198,[205][206][207] . Further, residual copper(II) ions may displace the far less abundant PET radiometals from their chelators 208 . In sharp contrast, secondgeneration AACs are catalyst-free and, hence, better tolerated by biomolecules 173 . Indeed, by employing ring strain-activated cyclic alkynes, such as dibenzocyclooctyne, the reaction can be accomplished in aqueous solutions, at ambient temperature and without copper catalyst 209,210 . This strain-promoted azide-alkyne cycloaddition (SPAAC) has been used for the radiofluorination of bombesin derivatives, RGD peptides, nanobodies and monoclonal antibodies 203,[211][212][213][214][215][216] . Despite its widespread use, the SPAAC ligation is limited by the formation of a lipophilic and bulky benzocyclooctatriazole footprint (Fig. 9b), which can lead to substantial changes in tracer pharmacokinetics 217 . In contrast, the traceless Staudinger ligation offers a useful alternative with a smaller and less lipophilic footprint and has been employed for the radiofluorination of several peptides [218][219][220][221][222] . Major limitations of the Staudinger ligation, however, encompass the lack of in vivo selectivity as well as the slow reaction kinetics at ambient temperature, which confines its utility to in vitro applications involving temperature-resistant vectors 223,224 .
Similar to the SPAAC and Staudinger ligations, the inverse electron demand Diels-Alder (IEDDA) cycloaddition does not require the use of a metal catalyst. The IEDDA reaction proceeds remarkably fast and orthogonal between tetrazine moieties and dienophiles-typically being either trans-cyclooctene or norbornene 225 . Given the fast reaction rate, IEDDA cycloadditions are particularly useful for labeling short-lived radionuclides 226 . Along this line of reasoning, this reaction has been used to label a number of peptides, including RGD, Exendin, glucagon-like peptide, and a PARP1 inhibitor analog [227][228][229][230] . Nonetheless, it should be noted that isomerization issues, as well as a lipophilic footprint, constitute drawbacks of the IEDDA. While the IEDDA has recently been translated to clinical testing by assessing its utility for drug delivery optimization of doxorubicin (NCT04106492), applications in combination with radiolabeled compounds are eagerly awaited.
Pre-targeting approaches. Although antibodies are highly promising vectors due to their excellent affinity and selectivity, they typically exhibit slow pharmacokinetics and biological half-lives of several days. Accordingly, they are only compatible with long-lived radionuclides. Of note, the sluggish clearance from blood circulation, alongside the slow radioactive decay, is associated with high background noise and radiation dose for the patient 231 . To circumvent this core limitation of radiolabeled antibodies, in vivo pre-targeting strategies were developed, whereby a functionalized antibody is injected some days prior to the radionuclide-bearing prosthetic group. Notably, the uncoupling of antibody and radionuclide injection permits the antibody vector to accumulate at the target site, while being cleared from the blood circulation, not only reducing radiation exposure to non-target organs but also improving signal-to-background ratios in regions of interest 176,232 . Once the antibody has largely been cleared from the blood circulation, the radionuclide-bearing prosthetic group is administered, thus allowing the bioconjugation reaction to occur in vivo 233,234 . In addition to the dosimetric benefit to non-target organs and reduced background uptake, pre-targeting with fast and efficient click-type chemistry has rendered antibodies compatible with the labeling with short-lived PET nuclides, including fluorine-18 [235][236][237][238] . A While peptides and proteins were traditionally labeled via NH 2 -and thiol-specific modification with N-hydroxysuccinimides (NHS), isothiocyanates, or maleimides, respectively, B modern (Click-type) bioconjugation strategies involve fast and high-yielding orthogonal radiochemistry with non-naturally occurring chemical functionalities. Created with BioRender.com.

Conclusion and perspectives
Recent breakthroughs in molecular imaging with PET have undoubtedly improved diagnostic procedures in clinical oncology, cardiology, and neurology. PET probes are increasingly employed for target engagement studies, thereby facilitating drug discovery and development. However, the rapid growth in nuclear medicine and drug discovery for radiopharmaceuticals poses inherent challenges to radiochemists, since the radiolabeling of many biologically relevant compounds remains difficult. As such, strenuous efforts have been devoted to the development of new radiosynthetic strategies, including the progress from the patents [259][260][261][262] . Over the past decade, these efforts provided a plethora of novel synthetic options and broadened the scope of functional groups that can be harnessed for radiolabeling. To date, the most advanced radiofluorination methods, allowing the labeling of both activated and non-activated arenes, include coppermediated reactions with boron/tin precursors and metal-free hypervalent iodine(III) methods. Similarly, 11 C-labeling through [ 11 C]CO 2 fixation-mediated carbonylation has been translated to human use and enabled the labeling of various functionalities, including carboxylic acids, amides, carbamates, oxazolidinones, and unsymmetrical ureas. With regard to the labeling of biomolecules, traditional bioconjugation methods that involve the functionalization of primary amines and thiols with NHS, isothiocyanates, anhydrides, or maleimides, respectively, are being well complemented by modern click-type bioconjugation methods such as azide-alkyne cycloadditions and inverse electron demand Diels-Alder reactions. The fast and high-yielding orthogonal radiochemistry with non-naturally occurring chemical functionalities makes the click-type bioconjugations methods particularly promising for future clinical applications. Further, concepts that promote in-house preparation of radiopharmaceuticals have been playing a key role in fostering innovation and enabling cutting-edge applications for patients in need 263 . It is worth mentioning that, attributed to the rapid evolution of PET chemistry, it is critical to follow the consensus nomenclature rules and good practice 33 in reporting radiochemistry discoveries, facilitating the comparison and translation of novel methods. Indeed, the remarkable advances in radiochemistry have led to a paradigm shift by enabling the design and synthesis of novel radiopharmaceuticals, thereby harnessing innovative concepts and promising approaches to radiotheranostics. In times when precision medicine has reshaped our healthcare system, exploiting such readily available concepts seems invaluable to improve patient management and therapeutic outcomes.